Anatomy and Neuroscience - Research Publications

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    Genetic control of the innate immune response.
    Wells, CA ; Ravasi, T ; Faulkner, GJ ; Carninci, P ; Okazaki, Y ; Hayashizaki, Y ; Sweet, M ; Wainwright, BJ ; Hume, DA (Springer Science and Business Media LLC, 2003-06-26)
    BACKGROUND: Susceptibility to infectious diseases is directed, in part, by the interaction between the invading pathogen and host macrophages. This study examines the influence of genetic background on host-pathogen interactions, by assessing the transcriptional responses of macrophages from five inbred mouse strains to lipopolysaccharide (LPS), a major determinant of responses to gram-negative microorganisms. RESULTS: The mouse strains examined varied greatly in the number, amplitude and rate of induction of genes expressed in response to LPS. The response was attenuated in the C3H/HeJlpsd strain, which has a mutation in the LPS receptor Toll-like receptor 4 (TLR4). Variation between mouse strains allowed clustering into early (C57Bl/6J and DBA/2J) and delayed (BALB/c and C3H/ARC) transcriptional phenotypes. There was no clear correlation between gene induction patterns and variation at the Bcg locus (Slc11A1) or propensity to bias Th1 versus Th2 T cell activation responses. CONCLUSION: Macrophages from each strain responded to LPS with unique gene expression profiles. The variation apparent between genetic backgrounds provides insights into the breadth of possible inflammatory responses, and paradoxically, this divergence was used to identify a common transcriptional program that responds to TLR4 signalling, irrespective of genetic background. Our data indicates that many additional genetic loci control the nature and the extent of transcriptional responses promoted by a single pathogen-associated molecular pattern (PAMP), such as LPS.
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    Alternate transcription of the Toll-like receptor signaling cascade
    Wells, CA ; Chalk, AM ; Forrest, A ; Taylor, D ; Waddell, N ; Schroder, K ; Himes, SR ; Faulkner, G ; Lo, S ; Kasukawa, T ; Kawaji, H ; Kai, C ; Kawai, J ; Katayama, S ; Carninci, P ; Hayashizaki, Y ; Hume, DA ; Grimmond, SM (BMC, 2006-01-01)
    BACKGROUND: Alternate splicing of key signaling molecules in the Toll-like receptor (Tlr) cascade has been shown to dramatically alter the signaling capacity of inflammatory cells, but it is not known how common this mechanism is. We provide transcriptional evidence of widespread alternate splicing in the Toll-like receptor signaling pathway, derived from a systematic analysis of the FANTOM3 mouse data set. Functional annotation of variant proteins was assessed in light of inflammatory signaling in mouse primary macrophages, and the expression of each variant transcript was assessed by splicing arrays. RESULTS: A total of 256 variant transcripts were identified, including novel variants of Tlr4, Ticam1, Tollip, Rac1, Irak1, 2 and 4, Mapk14/p38, Atf2 and Stat1. The expression of variant transcripts was assessed using custom-designed splicing arrays. We functionally tested the expression of Tlr4 transcripts under a range of cytokine conditions via northern and quantitative real-time polymerase chain reaction. The effects of variant Mapk14/p38 protein expression on macrophage survival were demonstrated. CONCLUSION: Members of the Toll-like receptor signaling pathway are highly alternatively spliced, producing a large number of novel proteins with the potential to functionally alter inflammatory outcomes. These variants are expressed in primary mouse macrophages in response to inflammatory mediators such as interferon-gamma and lipopolysaccharide. Our data suggest a surprisingly common role for variant proteins in diversification/repression of inflammatory signaling.
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    Genome-wide review of transcriptional complexity in mouse protein kinases and phosphatases
    Forrest, ARR ; Taylor, DF ; Crowe, ML ; Chalk, AM ; Waddell, NJ ; Kolle, G ; Faulkner, GJ ; Rimantas, K ; Katayama, S ; Wells, C ; Kai, C ; Kawai, J ; Carninci, P ; Hayashizaki, Y ; Grimmond, SM (BMC, 2006-01-01)
    BACKGROUND: Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse http://variant.imb.uq.edu.au. RESULTS: By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys (seven known and 19 novel: Alk, Csf1r, Egfr, Epha1, 3, 5,7 and 10, Ephb1, Flt1, Flt3, Insr, Insrr, Kdr, Met, Ptk7, Ptprc, Ptprd, Ptprg, Ptprl, Ptprn, Ptprn2, Ptpro, Ptprr, Ptprs, and Ptprz1) and 13 transmembrane forms (four known and nine novel: Axl, Bmpr1a, Csf1r, Epha4, 5, 6 and 7, Ntrk2, Ntrk3, Pdgfra, Ptprk, Ptprm, Ptpru). Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms. CONCLUSION: These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways.
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    Complex loci in human and mouse genomes
    Engstrom, PG ; Suzuki, H ; Ninomiya, N ; Akalin, A ; Sessa, L ; Lavorgna, G ; Brozzi, A ; Luzi, L ; Tan, SL ; Yang, L ; Kunarso, G ; ng, EL-CN ; Batalov, S ; Wahlestedt, C ; Kai, C ; Kawai, J ; Carninci, P ; Hayashizaki, Y ; Wells, C ; Bajic, VB ; Orlando, V ; Reid, JF ; Lenhard, B ; Lipovich, L ; Blake, J ; Hancock, J ; Pavan, B ; Stubbs, L (PUBLIC LIBRARY SCIENCE, 2006-04-01)
    Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.
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    Transcript annotation in FANTOM3: Mouse gene catalog based on physical cDNAs
    Maeda, N ; Kasukawa, T ; Oyama, R ; Gough, J ; Frith, M ; Engstrom, PG ; Lenhard, B ; Aturaliya, RN ; Batalov, S ; Beisel, KW ; Bult, CJ ; Fletcher, CF ; Forrest, ARR ; Furuno, M ; Hill, D ; Itoh, M ; Kanamori-Katayama, M ; Katayama, S ; Katoh, M ; Kawashima, T ; Quackenbush, J ; Ravasi, T ; Ring, BZ ; Shibata, K ; Sugiura, K ; Takenaka, Y ; Teasdale, RD ; Wells, CA ; Zhu, Y ; Kai, C ; Kawai, J ; Hume, DA ; Carninci, P ; Hayashizaki, Y (PUBLIC LIBRARY SCIENCE, 2006-04-01)
    The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.
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    A Cross-Study Transcriptional Analysis of Parkinson's Disease
    Sutherland, GT ; Matigian, NA ; Chalk, AM ; Anderson, MJ ; Silburn, PA ; Mackay-Sim, A ; Wells, CA ; Mellick, GD ; Cookson, MR (PUBLIC LIBRARY SCIENCE, 2009-03-23)
    The study of Parkinson's disease (PD), like other complex neurodegenerative disorders, is limited by access to brain tissue from patients with a confirmed diagnosis. Alternatively the study of peripheral tissues may offer some insight into the molecular basis of disease susceptibility and progression, but this approach still relies on brain tissue to benchmark relevant molecular changes against. Several studies have reported whole-genome expression profiling in post-mortem brain but reported concordance between these analyses is lacking. Here we apply a standardised pathway analysis to seven independent case-control studies, and demonstrate increased concordance between data sets. Moreover data convergence increased when the analysis was limited to the five substantia nigra (SN) data sets; this highlighted the down regulation of dopamine receptor signaling and insulin-like growth factor 1 (IGF1) signaling pathways. We also show that case-control comparisons of affected post mortem brain tissue are more likely to reflect terminal cytoarchitectural differences rather than primary pathogenic mechanisms. The implementation of a correction factor for dopaminergic neuronal loss predictably resulted in the loss of significance of the dopamine signaling pathway while axon guidance pathways increased in significance. Interestingly the IGF1 signaling pathway was also over-represented when data from non-SN areas, unaffected or only terminally affected in PD, were considered. Our findings suggest that there is greater concordance in PD whole-genome expression profiling when standardised pathway membership rather than ranked gene list is used for comparison.
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    Using a 3D virtual muscle model to link gene expression changes during myogenesis to protein spatial location in muscle
    Waardenberg, AJ ; Reverter, A ; Wells, CA ; Dalrymple, BP (BMC, 2008-10-22)
    BACKGROUND: Myogenesis is an ordered process whereby mononucleated muscle precursor cells (myoblasts) fuse into multinucleated myotubes that eventually differentiate into myofibres, involving substantial changes in gene expression and the organisation of structural components of the cells. To gain further insight into the orchestration of these structural changes we have overlaid the spatial organisation of the protein components of a muscle cell with their gene expression changes during differentiation using a new 3D visualisation tool: the Virtual Muscle 3D (VMus3D). RESULTS: Sets of generic striated muscle costamere, Z-disk and filament proteins were constructed from the literature and protein-interaction databases. Expression profiles of the genes encoding these proteins were obtained from mouse C2C12 cells undergoing myogenesis in vitro, as well as a mouse tissue survey dataset. Visualisation of the expression data in VMus3D yielded novel observations with significant relationships between the spatial location and the temporal expression profiles of the structural protein products of these genes. A muscle specificity index was calculated based on muscle expression relative to the median expression in all tissues and, as expected, genes with the highest muscle specificity were also expressed most dynamically during differentiation. Interestingly, most genes encoding costamere as well as some Z-disk proteins appeared to be broadly expressed across most tissues and showed little change in expression during muscle differentiation, in line with the broader cellular role described for some of these proteins. CONCLUSION: By studying gene expression patterns from a structural perspective we have demonstrated that not all genes encoding proteins that are part of muscle specific structures are simply up-regulated during muscle cell differentiation. Indeed, a group of genes whose expression program appears to be minimally affected by the differentiation process, code for proteins participating in vital skeletal muscle structures. Expression alone is a poor metric of gene behaviour. Instead, the "connectivity model of muscle development" is proposed as a mechanism for muscle development: whereby the closer to the myofibril core of muscle cells, the greater the gene expression changes during muscle differentiation and the greater the muscle specificity.